The pineal complex of the three-spined stickleback,Gasterosteus aculeatus L.

Summary The pineal complex of the three-spined stickleback (Gasterosteus aculeatus L.) was investigated by light and electron microscopy, as well as fluorescence histochemistry for demonstration of catecholamines and indolamines. The pineal complex of the stickleback consists of a pineal organ and a small parapineal organ situated on the left side of the pineal stalk. The pineal organ, including the entire stalk, is comprised mainly of ependymal-type interstitial cells and photoreceptor cells with well-developed outer segments. Both unmyelinated and myelinated nerve fibres are present in the pineal organ. Nerve tracts from the stalk enter the habenular and posterior commissures. A small bundle of nerve fibres connects the parapineal organ and the left habenular body. The presence of indolamines (5-HTP, 5-HT) was demonstrated in cell bodies of both the pineal body and the pineal stalk, and catecholaminergic nerve fibres surround the pineal complex.     

The Parapineal Organ of Teleosts

A parapineal organ was found to be present in 21 teleost fishes belonging to 20 different families, but was absent in poecilids and cyprinodontids. The parapineal organ was situated on the left side of the brain and sent a nerve tract to the left habenular nucleus, except in Gadus, where a “parapineal organ” appeared to send a nerve tract into the pineal stalk. The parapineal organ of adult Gasterosteus consisted of glial elements and parapinealocytes. The latter were small neurons which sent off the unmyelinated axons that formed the parapineal tract. A single photoreceptor cell was found in a stickleback parapineal organ.     

The parapineal and pineal organs of the elver (glass eel), Anguilla anguilla L.

Summary The parapineal organ of the glass eel (elver) consists of approximately 400 cells and is situated to the left of the connection of the pineal stalk to the third ventricle. A conspicuous nerve tract containing approximately 350 fibers arises from the parapineal organ and runs in spatial relationship to the habenular commissure toward the left habenular nucleus. The dominating cell type of the parapineal organ of the elver is a neuron (sensory neuron) of small diameter provided with atypical cilia (9×2+0, or rarely 8×2+0 types). Well-developed photoreceptor outer segments are lacking, and no interstitial cells of ependymal type have been observed with certainty in the parapineal organ. The axonal processes from the nerve cells form the tract leaving the parapineal organ.The pineal organ proper of the elver consists of photoreceptor cells with well-developed outer segments, interstitial cells of ependymal type, and ganglion cells. Axons from the latter form the pineal tract, which leaves the pineal organ and runs in close contact with the subcommissural organ toward the posterior commissure. The proximal part of the pineal stalk contains only a few photoreceptor cells the outer segments of which are less developed than those of the pineal body and the distal part of the pineal stalk.     

Immunocytochemical localization of serotonin and photoreceptor-specific proteins (rod-opsin,S-antigen) in the pineal complex of the river lamprey,Lampetra japonica,with special reference to photoneuroendocrine cells

Summary The pineal complex of the river lamprey, Lampetra japonica, was examined by means of immunocytochemistry with antisera against serotonin, the precursor of melatonin, and two photoreceptor proteins, rod-opsin (the apoprotein of the photopigment rhodopsin) and S-antigen. Serotonin-immunoreactive cells were observed in both the pineal and the parapineal organ. The proximal portion of the pineal organ (atrium) comprised numerous serotonin-immunoreactive cells displaying spherical somata. In the distal end-vesicle of the pineal organ, the serotonin-immunoreactive elements resembled photoreceptors in their size and shape. These cells projecting into the pineal lumen and toward the basal lamina were especially conspicuous in the ventral portion of the end-vesicle. In addition, single serotonin-immunoreactive nerve cells were found in this location. Retinal photoreceptors were never seen to contain immunoreactive serotonin; amacrine cells were the only retinal elements exhibiting serotonin immunoreaction. Strong S-antigen immunoreactivity was found in numerous photoreceptors located in the pineal end-vesicle. In contrast, the S-antigen immunoreactivity was weak in the spherical cells of the atrium. Thus, the pattern of S-antigen immunoreactivity was roughly opposite to that of serotonin. Similar findings were obtained in the parapineal organ. The rod-opsin immunoreaction was restricted to the outer segments of photoreceptors in the pineal end-vesicle and parapineal organ. No rodopsin immunoreactive outer segments occurred in the proximal portion of the atrium. Double immunostaining was employed to investigate whether immunoreactive opsin and serotonin are colocalized in one and the same cell. This approach revealed that (i) most of the rodopsin-immunoreactive outer segments in the end-vesicle belonged to serotonin-immunonegative photoreceptors; (ii) nearly all serotonin-immunoreactive cells in the end-vesicle bore short rod-opsin-immunoreactive outer segments protruding into the pineal lumen; and (iii) the spherical serotonin-immunoreactive cells in the pineal stalk lacked rod-opsin immunoreaction and an outer segment. These results support the concept that multiple cell lines of the photoreceptor type exist in the pineal complex at an early evolutionary stage.     

Substance P-like-immunoreactive neurons in the photosensory pineal organ of the rainbow trout,Salmo gairdneri Richardson (Teleostei)

Summary Restricted numbers of substance P-like-immuno-reactive (SPL-IR) neurons were demonstrated in the photosensory pineal organ of the rainbow trout. The small parapineal organ of this teleost species receives a distinct SPL-IR innervation via the habenular nuclei, but displays no intrinsic SPL-IR neurons. Intrapineal SPL-IR neurons were located in the rostral portion of the pineal end-vesicle. Neuronal somata were found in a lateral position with smooth axonal processes extending mediad. Immunoreactive somata and axonal processes were observed intraparenchymally as well as in the pineal lumen. The pattern of immunoreactivity was not changed in excised pineal organs that had been incubated in tissue culture medium in the dark for 18 h. The possibility that the intrapineal SPL-IR neurons are not part of the neural circuitry involved in the transduction of photic information, but may have other functions, is discussed.Fellow of the Alexander von Humboldt-Stiftung, Bonn, Federal Republic of GermanySupported by research funds from the Deutsche Forschungsge-meinschaft (Ko 758/2-4)     

Opsin-immunoreactive outer segments and acetylcholinesterase-positive neurons in the pineal complex of Phoxinus phoxinus (Teleostei,Cyprinidae)

Summary The pineal complex of the teleost, Phoxinus phoxinus L., was studied light-microscopically by the use of the indirect immunocytochemical antiopsin reaction and the histochemical acetylcholinersterase (AChE) method.Opsin-immunoreactive outer segments of photoreceptor cells were demonstrated in large numbers in all divisions of the pineal end-vesicle and in the pineal stalk. Moreover, they were found in the roof of the third ventricle, adjacent to the orifice of the pineal recess as well as scattered in the parapineal organ. These immunocytochemical observations provide direct evidence of the presence of an opsin associated with a photopigment in the photosensory cells of the pineal and parapineal organs of Phoxinus. By means of the AChE reaction (Karnovsky and Roots 1964) inner segments of pineal photoreceptors, intrinsic nerve cells, several intrapineal bundles of nerve fibers, and a prominent pineal tract were specifically marked. The pineal neurons can be divided into two types: one is located near the pineal lumen, the other near the basal lamina. The latter perikarya bear stained processes directed toward the photoreceptor layer. A rostral aggregation of two types of AChE-positive nerve cells occurs in the ventral wall of the pineal end-vesicle. The main portion of the AChE-positive pineal tract, which lies within the dorsal wall of the pineal stalk, can be followed to the posterior commissure where some of the nerve fibers course laterally. A few AChE-positive pineal nerve fibers run toward the lateral habenular nucleus via the habenular commissure. In the region of the subcommissural organ single AChE-positive neurons accompany the pineal tract. The nerve cells of the parapineal organ exhibit a moderate AChE activity.These findings extend the structural basis for the remarkable light-dependent activity of the pineal organ of Phoxinus phoxinus. To the memory of Professor Karl von Frisch, pioneer and master in the field of photoneuroendocrine systemsThis investigation was supported by grants from the Deutsche Forschungsgemeinschaft to A.O. (Ok 1/24; 1/25: Mechanismen biologischer Uhren) and to H.-W. K. (Ko 758/1; 758–2)On leave from the 2nd Department of Anatomy, SOTE, Budapest, Hungary     

The parapineal mediates left-right asymmetry in the zebrafish diencephalon

The dorsal diencephalon (or epithalamus) of larval zebrafish displays distinct left-right asymmetries. The pineal complex consists of the pineal organ anlage and an unpaired, left-sided accessory organ - the parapineal. The neighboring brain nuclei, the left and right dorsal habenulae, show consistent differences in their size, density of neuropil and gene expression. Mutational analyses demonstrate a correlation between the left-right position of the parapineal and the laterality of the habenular nuclei. We show that selective ablation of the parapineal organ results in the loss of habenular asymmetry. The left-sided parapineal therefore influences the left-right identity of adjacent brain nuclei, indicating that laterality of the dorsal diencephalon arises in a step-wise fashion.     

Structure of the parapineal organ of the adult rainbow trout,Salmo gairdneri Richardson

Summary The parapineal organ of the teleost Salmo gairdneri Richardsonsu1 was investigated with the light and electron microscopes. It is a small cell mass, 0.1–0.3 mm in diameter, containing a narrow lumen and consistently situated to the left of the pineal stalk and dorsal to the left habenular nucleus. It is connected with the habenular nucleus through a conspicuous parapineal tract. The parapineal organ continues to grow at least until the fish reaches sexual maturity and shows no sign of cellular degeneration at the age of two years.The parapineal tissue consists of supporting cells and nerve cells; the latter give rise to the axons of the parapineal tract. Furthermore, a small number of receptor cells of the type existing in the pineal organ is present. No morphological evidence was obtained to suggest a sensory or secretory function of the parapineal organ.The existence of the parapineal organ in the adult pike, Esox lucius, L., and of a connection between the pineal tract and the habenular commissure in Salmo gairdneri is briefly reported. The results are discussed in the light of existing literature.Work done with the aid of a research scholarship from the Alexander von Humboldt Foundation, Bad Godesberg, Germany. —The electron microscope used in this study was placed at the disposal of Prof. Oksche by the Deutsche Forschungsgemeinschaft. —I wish to thank Prof. Oksche for the facilities made available at his institute and for his helpful interest in my work.     

Studies on the subcommissural organ of Salmo gairdneri

The light microscopic analysis of serial sections of the subcommissural organ (SCO) of the rainbow trout (Salmo gairdneri) shows that the form of the groove-like (in cross section) organ varies over its total length. Its rostral origin is a tunnel-like structure anterior to the orifice of the hollow pineal stalk. The SCO forms the dorsal wall of the brain. Caudally the SCO is increasingly displaced from the surface of the brain by the fibers of the posterior commissure; the organ ends in a tabular area beyond the latter. The orifice of the pineal stalk is surrounded by the ependyma of the SCO that invaginates like a funnel and joins with the ependyma of the pineal stalk after a considerable narrowing. The rudimentary parapineal organ is located on the left side of the brain and is connected with the left habenular ganglion through the parapineal tract. It contacts the third ventricle with a short channel within the ependyma of the SCO. The histological organization of the ependymal and hypendymal cells of the SCO is typical of teleosts. Secretory material is located basally and apically in relation to the nucleus, but there is no indication of a basal secretory release. Basal ependymal processes terminate with broadened endings at the membrana limitans externa. The apical product is discharged into the third ventricle, where it aggregates into the thread-like structure of Reissner's fibre. The SCO cells have no direct contact with cerebral or meningeal blood vessels.     

Photoreceptor-like outer segments in the pineal organ of the lovebird,Uroloncha domestica (Aves: Passeriformes)

Summary In the pineal organ of the lovebird, Uroloncha domestica, bulbous, cup-shaped and elongated outer segments of photoreceptor-like pinealocytes are demonstrated by scanning electron microscopy. These scarce outer segments, 4–11 m in length, extend into the pineal lumen. The present structural observations speak in favor of photosensitive pinealocytes in the pineal organ of Uroloncha domestica. The relation of the photoreceptor-like pinealocytes to acetylcholinesterase-positive nerve cells and a nervous connection between the pineal and the brain indicate that the pineal organ of this passeriform species may be the site of neuroendocrine and photoreceptive functions.Supported by a fellowship from the Japan Society for the Promotion of Science to M. UeckSupported by a grant from the Ministry of Education of Japan to K. Wake and by a grant of the Deutsche Forschungsgemeinschaft to M. Ueck     

Photoreceptor cells and neural elements with long axonal processes in the pineal organ of the lamprey,Lampetra japonica,identified by use of the horseradish peroxidase method

Summary Horseradish peroxidase (HRP) was applied to the transected end of the pineal tract of the lamprey, Lampetra japonica. Distinct reaction products of HRP were observed in 2 types of cell other than ganglion cells. The first type of cell protrudes a knob-like process into the pineal lumen. This type of cell was clearly identified by electron microscopy as a photoreceptor cell; its outer segment was connected to the ellipsoid through a sensory cilium. The other type of cell was located among photoreceptor and supporting cells. The processes of these cells were thin and slender, and they obviously did not represent photoreceptor, supporting, or conventional ganglion cells. The present results indicate that, in the lamprey, some of the photoreceptor cells of the pineal organ project their axon-like processes toward the posterior commissure, but that there is also another type of cell displaying long axonal projections. HRP-containing cells were distributed randomly over the pineal organ and were occasionally also observed in the parapineal organ.     

Photoreceptor development in the pineal organ and the eye of Plecoglossus altivelis and Paralichthys olivaceus (Teleostei)

Summary The ontogenetic apperance of pineal photo-receptors was compared with that of retinal photoreceptors in the ayu Plecoglossus altivelis and the lefteye flounder Paralichthys olivaceus, which hatched 10 days and 3 days after fertilization, respectively. Despite the disparity in incubation time, the outer segments (containing membranous lamellae) of the pineal photoreceptors first appeared from 3 to 4 days after fertilization in both species. In contrast, the outer segments of the retinal photoreceptors first became visible 5 to 6 days after fertilization, although a characteristic retinal stratification and the optic tract leaving the ganglion cell layer were already found 4 days after fertilization in both species. The functional significance of these temporal disparities and/or similarities in photoreceptor development are discussed with special reference to the timing of daily rhythmic activities during the early developmental period of the teleosts.The results were presented at the Annual Meeting of the Japanese Society of Scientific Fisheries on April 2, 1990 (Tokyo)     

Development of the innervation in the human pineal organ

Summary This investigation is concerned with pineal organs of human embryos 60 to 150 days old. At every stage central nerve fibres enter the pineal organ by way of the habenular commissure, but are restricted to the pineal's proximal part. On about the 60th day of the development the sympathetic nervus conarii grows into the distal pole of the pineal organ from a dorso-caudal direction and plays the predominant part in the innervation of the pineal organ. After penetrating, it soon branches out and forms a network in the pineal tissue. Much later, not until the 5th embryonic month, sympathetic nerves appear accompanying the supplying vessels in the perivascular spaces. After a short time these nerves pierce the outer limiting basement membrane and penetrate the parenchyma. Towards the end of the 5th embryonic month the axons of the sympathetic nerves form varicosities containing clear and dense core vesicles. At this point large amounts of laminated granules appear primarily in cell processes, probably of pinealocytes. Isolated granules also occur in the varicosities of axons. The granules encountered here are most likely secretory granules.Dedicated to Professor Bargmann on his 65th birthday.     

Comparative ultrastructural investigations of the pineal organ of the blind cave fish,Anoptichthys jordani,and its ancestor,the eyed river fish,Astyanax mexicanus

A comparative ultrastructural study has been made of the pineal organ in specimens of two closely related populations of the characid fish, Astyanaz mexicanus. The specimens of one population are living in the river, under natural light conditions. The specimens of the other population, originally described as Anoptichthys jordani, are living in a completely dark cave. In specimens of both populations the pineal organ consists of a spindle shaped end-vesicle, connected to the diencephalic roof by a slender stalk. The pineal tissue is compact and consists predominantly of glia-like supporting cells and sensory cells resembling the photoreceptor cells of the lateral vertebrate eye. Phagocytotic microglia-like cells can be found in close contact with the outer segments of the sensory cells. Nerve cells are located in the neighbourhood of neuropil formations, in which synaptic contacts are established between sensory cells and nerve cells. From these nerve cells fibers are emerging, forming the pineal tract that runs down the pineal stalk towards the diencephalon. On the basis of the ultrastructure described by other authors it is concluded that the pineal organ in specimens of the river population of Astyanax mexicanus resembles the pineal organ of other fish species. In specimens of the river population, reared under normal light-dark conditions for 3, 9 or 18 months, conspicuous morphological changes have not been detected in the presumably light-sensitive outer segments of the sensory cells or in other parts of the pineal tissue. In specimens of the cave populations, reared under identical conditions, an age-dependent, gradual regression of the regular outer segment organization of the pineal sensory cells takes place. In other parts of the pineal tissue, only small morphological changes can be observed. In specimens of the cave population, reared in constant darkness, the regression of the pineal outer segment organization begins earlier and is obvious. It is postulated that the gradual age-dependent regression of the regular organization of the outer segments in the pineal organ of cave specimens of Astyanax mexicanus is genetically determined and indicates a regressive evolution of the pineal light sensitivity. The expression of the regressive traits is dependent on the environmental light conditions.     

Immunocytochemical localization of vitamin A in the retina and pineal organ of the frog, Rana esculenta

Vitamin A immunoreactive sites were studied in the retina and pineal organ of the frog, Rana esculenta, by the peroxidase antiperoxidase, avidin-biotinperoxidase and immunogold methods. In dark-adapted material, strong immunoreaction was found in the outer and inner segments of the photoreceptor cells of both retina and pineal organ, as well as in the pigment epithelium, retinal Müller cells and pineal ependymal cells. In light-adapted retina, cones and green (blue-sensitive) rods were immunopositive. At the electron microscopic level, immunogold particles were found on the membranes of the photoreceptor outer segments as well as on the membranes of the endoplasmic reticulum and mitochondria. Individual retinal photorecptor cells exhibited strong immunoreaction in the distal portion of the inner segment, the ciliary connecting piece and the electron-dense material covering the outer segment. In the pigment epithelium, the immunolabeling varied in intensity in the basal and apical cytoplasm and phagocytosed outer segments. The immunocytochemical results indicate that retinoids (retinal, retinol and possibly retinoic acid) are present not only in the photoreceptor cells of the retina but also in those of the pineal organ. The light-dependent differences in the immunoreactivity of vitamin A underlines its essential role in the visual cycle of the photopigments. Our results suggest that the pineal ependyma plays a role comparable to that of the Müller cells and pigment epithelium of the retina with regard to the transport and storage of vitamin A. The presence of a retinoid in nuclei, mitochondria and cytoplasmic membranes suggests an additional role of vitamin A in other metabolic processes.     

Building an asymmetric brain: Development of the zebrafish epithalamus

The human brain exhibits notable asymmetries. Little is known about these symmetry deviations; however scientists are beginning to understand them by employing the lateralized zebrafish epithalamus as a model. The zebrafish epithalamus consists of the pineal and parapineal organs and paired habenular nuclei located bilateral to the pineal complex. While zebrafish pineal and parapineal organs arise from a common population of cells, parapineal cells undergo a separate program that allows them to migrate left of the pineal anlage. Studying the processes that lead to brain laterality in zebrafish will allow a better understanding of how human brain laterality is established.     

The pineal organ as a folded retina: immunocytochemical localization of opsins

The most simple pineal complex (the pineal and parapineal organs of lampreys), consists of saccular evaginations of the diencephalic roof, and has a retina-like structure containing photoreceptor cells and secondary neurons. In more differentiated vertebrates, the successive folding of the pineal wall multiplies the cells and reduces the lumen of the organ, but the pattern of the histological organization remains similar to that of lampreys; therefore, we consider the histological structure of the pineal organ of higher vertebrates as a 'folded retina'. The cell membrane of several pineal photoreceptor outer-segments of vertebrates immunoreact with anti-retinal opsin antibodies supporting the view of retina-like organization of the pineal. Some other pineal outer segments do not react with retinal anti-opsin antibodies, a result suggesting the presence of special pineal photopigments in different types of pinealocytes that obviously developed during evolution. The chicken pinopsin, detected in the last years, may represent one of these unknown photopigments. Using antibodies against chicken pinopsin, we compared the immunoreactivity of different photoreceptors of the pineal organs from cyclostomes to birds at the light and electron microscopic levels. We found pinopsin immunoreaction on all pinealocytes of birds and on the rhodopsin-negative large reptilian pinealocytes. As the pinopsin has an absorption maximum at 470 nm, these avian and reptilian immunoreactive pinealocytes can be regarded as green-blue light-sensitive photoreceptors. Only a weak immunoreaction was observed on the frog and fish pinealocytes and no reaction was seen in cyclostomes and in the frontal organ of reptiles. Some photoreceptors of the retina of various species also reacted the pinopsin antibodies, therefore, pinopsin must have certain sequential similarity to individual retinal opsins of some vertebrates.     

GABAergic system of the pineal organ of an elasmobranch (Scyliorhinus canicula): a developmental immunocytochemical study

The present immunocytochemical study provides evidence of a previously unrecognized, rich, γ-aminobutyric acid (GABA)-ergic innervation of the pineal organ in the dogfish (Scyliorhinus canicula). In this elasmobranch, the pineal primordium is initially detected at embryonic stage 24 and grows to form a long pineal tube by stage 28. Glutamic acid decarboxylase (GAD)-immunoreactive (-ir) fibers were first observed at stage 26, and by stage 28, thin GAD-ir fibers were detectable at the base of the pineal neuroepithelium. In pre-hatchling embryos, most fibers gave rise to GAD-ir boutons that were localized in the basal region of the neuroepithelium, although a smaller number of labeled terminals ascended to the pineal lumen. A few pale GAD-ir perikarya were observed within the pineal organ of stage 29 embryos, but GAD-ir perikarya were not observed at other developing stages or in adults. In contrast, GABA immunocytochemistry revealed the presence of GABAergic perikarya and fibers in the pineal organ of late stage embryos and adults. Although high densities of GABAergic cells were observed in the paracommissural pretectum, posterior tubercle, and tegmentum of dogfish embryos (regions previously demonstrated to contain pinealopetal cells), the presence of GABA-ir perikarya in the pineal organ strongly suggests that the rich GABAergic innervation of the elasmobranch pineal organ is intrinsic. This contrasts with the central origin of GABAergic fibers in the pineal gland of some mammals. This work was supported by the Spanish Education and Science Ministry and FEDER (BXX2000-0453-C02 and BFU2004-03313/BF1), the Xunta de Galicia (PGIDT99BIO20002), and NIH/NIDCD awards R01 DC01705 and P01 DC01837 (to G.R.H.).     

Acetylcholinesterase-containing nerve cells and their distribution in the pineal organ of the goldfish,Carassius auratus

Summary Histochemically, an intense acetylcholinesterase (AChE) reaction has been observed in the perikarya of the nerve cells and in the neuropil formations of the pineal organ in the goldfish, Carassius auratus. A group of AChE-rich nerve cells has also been observed between the caudal end of the pineal stalk and the habenular ganglion. No component of the complex revealed butyrylcholinesterase (BuChE) activity.Two different types of nerve cells were recognized on the basis of their size, AChE activity and distribution. Type I cells are characterized by large perikarya possessing a moderate AChE activity and by the presence of an extensive AChE-rich neuropil formation in their vicinity; they are restricted to the rostro-lateral regions of the pineal vesicle. Type II cells are situated in the medio-rostral area of the pineal vesicle and along the entire length of the stalk, and are smaller than Type I cells; they show an intense AChE activity in their perikarya.The neuropil formations in the medio-rostral area of the pineal vesicle are almost as large as those in the vicinity of the Type I cells; they exhibit a strong AChE activity. In the rostral half of the vesicle several sensory cells are associated with each nerve cell, while in the caudal portion only a few cells are apposed to each nerve cell. Thus, the ratio of the number of sensory cells to that of AChE-containing nerve cells in the anterior half of the pineal vesicle is high when compared with the remaining area. In the anterior half of the vesicle the outer segments of the sensory cells are more distinct and their inner segments possess a higher AChE activity than those in the posterior region and the stalk. A gradation in the degree of development of neuropil formations along the pineal axis is remarkable; their size and AChE activity gradually diminish in a caudal direction. In view of the structural specialization of the rostral region of the pineal organ, it has been argued that its terminal portion is more photosensitive.This work was supported by a fellowship from the Alexander von Humboldt Foundation, Federal Republic of Germany.